Project Summary/Abstract Craniofacial malformations are among the most common human birth defects, affecting 1/700 births. While many of the genes that underlie these defects are known, the identity and functions of many others are not. The overarching goal of this proposal is evaluate the role of a largely uncharacterized gene, limb bud and heart homolog (lbh), during zebrafish craniofacial development. This gene may mediate CHARGE syndrome, a disease of neural crest cells (NCCs) that includes craniofacial defects such as micrognathia. Additionally, we have associated this gene with the evolution of jaws in cichlids, an evolutionary model that exhibits extensive diversity in craniofacial form, many of which mimic human facial malformations. Combined, these data led to the hypothesis that lbh is a novel regulator of NCC and craniofacial development, which will be tested through the following experimental aims. Aim 1: Given the paucity of information about lbh, we will first characterize the expression pattern and cellular specificity of lbh throughout zebrafish facial development using whole-mount and sectioned in situ hybridization and comparison with known craniofacial markers. Aim 2: We will evaluate the effects of modulating lbh expression (morpholino knockdown or mRNA overexpression) on the craniofacial skeleton. Preliminary experiments indicate that depletion of Lbh results in reduction of NCCs and commensurate defects in the craniofacial skeleton. Specifically, we will determine if the cellular mechanism of this defect is due to a failure of induction, failure of migration, decreased survival, or premature differentiation of NCCs using a combination of in situ hybridization, reporter zebrafish strains, and cellular assays. Aim 3: We will analyze the functional conservation and evolution of lbh using biologically relevant alleles of lbh isolated from cichlids with differing mandible lengths. Overall, these studies will illuminate the role of lbh in craniofacial development, disease, and evolution.